Enzymatic Synthesis of γ‐Glutamyl Dipeptides Catalysed by Mutant E. coli γ‐Glutamyltransferases

• Published in: European journal of organic chemistry 2022-11, Vol.2022 (43), p.n/a
• Main Authors: Rabuffetti, Marco, Speranza, Giovanna, Calvio, Cinzia, Morelli, Carlo F.
• Format: Article
• Language: English
• Subjects: Amino acids; Binding sites; Biocatalysis; Biological properties; Chemical synthesis; E coli; Mutagenesis; Residues; Substrates; Taste-active compounds; γ-Glutamyltransferases
• ISSN: 1434-193X; 1099-0690
• DOI: 10.1002/ejoc.202200907

γ‐Glutamyltransferases (GGTs) from different sources have been proposed in recent times as biocatalysts for the enzymatic synthesis of naturally occurring γ‐glutamyl derivatives with flavor‐enhancer properties and interesting biological activities. Although the enzymatic approach is considered as a viable alternative to both the troublesome and low‐yielding extraction from natural sources and synthesis through peptide chemistry requiring protection/deprotection steps, yields are not completely satisfactory, due to the intervention of GGT‐catalysed hydrolysis and autotranspeptidation side‐reactions. Here, the design and the use as biocatalyst for preparative purposes of two mutants of E. coli GGT are described. The design of mutants was pursued by docking‐guided identification of residues putatively involved in interaction with the acceptor substrate, thus probably representing a first identification of residues constituting the still elusive and poorly characterized acceptor substrate binding site of the enzyme. Two residues were identified inside the active site of E. coli γ‐glutamyltransferase, putatively involved in acceptor substrate binding. Point‐mutation of these residues afforded two mutant enzymes with altered catalytic properties. Mutant T413L showed a very promising transpeptidation‐to‐hydrolysis ratio up to 20 : 1. The two mutants were tested as biocatalysts for the enzymatic synthesis of γ‐glutamyl dipeptides with flavor‐enhancer properties.